Despite the fact that several alternative approaches such as microinjection into the cells, macroinjection or bombardment with DNA coated particles of the intact or cultured tissues have been claimed as methods for genetic transformation of crop plants, according to the available experimental data protoplasts are ideal and the most widely used objects for parasexual genetic manipulation methods including DNA transformation and somatic hybridization. These genetic manipulations require a methodology for protoplast isolation and culture. For practical applications obtaining fertile regenerants from the manipulated protoplasts is a basic prerequisite. Among the monocotyledonous crop species maize is one of the most studied tissue cultures. Several publications have shown that the potential for plant regeneration from 20 various cultured organs including anthers and immature embryoids is highly genotype-dependent (Green and Phillips, Crop Sci. 15, 417 (1975), Morocz, Tag-Ber., Acad. Landwirtsch.-Wiss. DDR, Berlin (1983), Duncan et al., Planta 165, 322 (1985); each of which is incorporated herein by reference). Improvements of culture conditions can only partially overcome these limitations (Duncan et al., Planta 165, 322 (1985)).
Dicotyledon plants can essentially be transformed via Tiplasmid vector systems with the aid of Agrobacterium tumefaciens. However, this system cannot be applied readily to monocotyledon plants. Potrykus et al. (Mol. Gen. Genet, 199, 183 (1985); incorporated herein by reference) and Lorz et al. (Mol. Gen. Genet, 199, 178 (1985); incorporated herein by reference) showed that plant protoplasts of monocotyledon plants can stably integrate foreign DNA into their genome. However, progress was first inhibited by the fact that the protoplasts could not be regenerated into fertile plants.
Over recent years there has been extensive research into the development of genotypes and processes to get the problem of plant regeneration under control. EP 29 24 435, incorporated herein by reference, relates to a process in which fertile plants can be obtained starting from a relatively non-mucilaginous, friable and granular maize callus. Shillito et al. (Bio/Technology 7, 581 (1989); incorporated herein by reference) have observed in this context that it is furthermore necessary for the regenerability into fertile plants to start from callus suspension cultures from which a dividing protoplast culture can be prepared which has the capability of regenerating plants. However, in this case, regenerable protoplasts could be isolated only after a long in vitro culture period of 7 to 8 months. Furthermore, the regenerated plants show abnormalities in morphology and fertility.
Prioli and Sbndahl (Bio/Technology 7, 589 (1989); incorporated herein by reference) relates to the regeneration and production of fertile plants from maize protoplasts of the Cateto maize inbred line Cat 100-1. The authors assume that protoplast regeneration into fertile plants is a function of a number of various factors, for example genotype, physiological state of the donor cells and the culture conditions. However, in this case, regenerable protoplasts could be isolated only after a long in vitro culture period of 20 to 40 months. Furthermore, the regenerated plants also show abnormalities with regard to morphology and reproductivity.
Thus, it would be advantageous to develop a new Zea mays (L.) genotype which can produce tissue and cell suspension cultures with the reproducible capacity to regenerate stably plants which are normal and predominantly fully fertile.